A sandwich-type electrochemical immunosensor for CRP detection using electrodeposited SWCNT/AuNP hybrid and AgNP-tagged VHH nanobody for signal amplification

C-reactive protein (CRP) is a highly sensitive inflammatory biomarker that rises significantly during inflammation caused by injury or infection; consistently elevated levels are also strongly associated with the progression of cardiovascular diseases (CVDs). In this study, we demonstrate a portable electrochemical sandwich immunosensor for CRP detection in human serum using an electrodeposited AuNP-wrapped SWCNT hybrid. The SWCNT network serves as a high-surface-area scaffold for the dense nucleation of AuNPs, which creates a synergistic effect that enhances the electrical conductivity and electrocatalytic activity of the carbon electrode. The nanohybrid (SWCNT/AuNP) facilitates the formation of a sandwich immunocomplex using capture IgG antibodies and AgNP-tagged VHH nanobodies as a signal amplifier. The compact size and stable orientation of VHH nanobodies minimize steric hindrance, allowing high-density signaling AgNP tags in close proximity to the electrode interface which significantly amplifies the electrochemical signal and improves the sensitivity. The electrochemical response (using amperometry and differential pulse voltammetry technique (DPV)) of the sandwich immunosensor was investigated across a wide range of CRP concentrations from 1 nM to 2 μM in buffer and diluted human serum. The immunosensor demonstrated a low limit of detection (LoD) of ∼287 pM (amperometry) and ∼841 pM (DPV) in buffer. The selectivity of the immunosensor was evaluated against common cardiac markers including troponins (cTnI and cTnT), myoglobin and BSA protein. Furthermore, the immunosensor exhibited good reproducibility and long-term stability for up to 3 weeks at 4 °C and under ambient conditions. The developed sandwich immunosensing strategy has the potential to be used in clinical setting for the rapid and early detection of acute inflammatory diseases. • A high-performance immunosensor was developed using a synergistic SWCNT/AuNP nanohybrid for enhanced electron transfer. • AgNP-tagged VHH nanobodies were employed as powerful signaling probes to achieve significant signal amplification. • The immunosensor demonstrated a wide dynamic range and low detection limits for CRP detection in diluted human serum. • The high selectivity and long-term stability highlight the future potential of this sandwich strategy for portable and PoC diagnostic applications.